Enhanced insect resistance in plants genetically engineered with a plant hormone gene involved in cytokinin biosynthesis

ABSTRACT

A transgenic plant into which a chimeric gene comprising a wound inducible promoter and a gene for an enzyme involved in cytokinin biosynthesis has been introduced shows enhanced resistance to insect infestation.

This is a continuation of application Ser. No. 08/054,985 filed Apr. 30,1993, now U.S. Pat. No. 5,496,732.

BACKGROUND OF THE INVENTION

1. Field of the Invention

One of the most important constraints on the yields of food and cashcrops worldwide can be attributed to insect attack. Based on 1987figures approximately 37% of all crops produced worldwide are lost topests such as insects (13%), disease (12%), and weeds and grasses (12%).Annually, large sums of money are spent on chemical pesticides to reducethese levels of crop damage. In 1987 the insecticide expenditures forthe three crops receiving the highest insecticide input--cotton, maizeand rice--was almost $4000 million. Approximately $3000 million wasspent on purchasing the insecticide and $1000 million for application.In the U.S. alone, over $400 million is spent each year for control oflepidopterans.

Dependance on chemical pesticides for crop damage control is not onlyexpensive, but it is also detrimental to the environment and unhealthyfor the animal population. Many chemical insecticides, particularlyorganophosphates and carbamates, are neurotoxic to a wide range ofanimals from honey bees to humans. A number of them have beendiscontinued because of their toxic properties. Thus, scientists arecurrently seeking alternatives to the conventional chemical approach tocrop pest management, and one approach is the investigation ofplant-mediated, and thus more environmentally friendly, methods andproducts.

Scientists have long used cross-breeding and hybridization techniques toprovide plants having particular desired traits such as increasedhardiness, nutritional value, taste, appearance, etc., but thesetechniques are at best lengthy, time-consuming processes which do notnecessarily result in the achievement of a particular goal. The adventof genetic engineering, however, provided the opportunity to introducegenetic material directly into a plant, which, upon expression in theplant, would result in a desired effect.

DESCRIPTION OF THE PRIOR ART

A limited number of insect-control agents are currently available forgenetic engineering into plants. The protein delta endotoxins from themicroorganism Bacillus thuringiensis (Bt) have been the most widelystudied in transformed plants, and the class of proteins known asproteinase inhibitors, when present at relatively high levels in thediet, has been shown to be effective against certain insects. Thepotential disadvantages to using transgenic Bt plants are that effectiveconcentrations may be difficult to achieve in the plant and that insectresistance may develop with time. The high levels of protein requiredfor insect killing and the potential need to target protein expressionto specific plant organs are problems associated with usage ofproteinase inhibitors as insect-control agents.

Phytohormones are known to have pivotal roles in promoting normal growthand development of plants and may also contribute to the mechanisms ofdefense (Gatehouse, 1991; Nicholson, 1992). Cytokinins are among themost active plant substances discovered and have been implicated in thephysiological and biochemical processes with marked effects onflowering, fruit set and ripening, leaf senescence, seed germination andstomatal function. Exogenously applied cytokinins have been shown tosuppress the induction of hypersensitive necrosis by viruses (Bailiss etal., 1977; Balazs and Kiraly, 1981). High endogenous cytokinin levels innon-rooting tobacco shoot lines (T-cyt) transformed with a gene involvedin cytokinin biosynthesis caused an increase in the expression ofdefense-related mRNAs (Memelink et al., 1987). A group of pathogenesisrelated proteins encoded by these genes is coordinately induced bywounding and pathogenic infections (Chen and Varner, 1985; Ward et al.,1991).

Cytokinins as well as other plant hormones have commercial applicationsas bioregulators and, in combination with endogenous hormones, mayprotect plants from pests and pathogens by inducing physiologicalchanges in the plants (Hallahan et al., 1991; Hedin et al., 1988; Thomasand Balkesley, 1987).

Cytokinins have also been shown to influence secondary metabolicpathways whose products exhibit insecticidal properties (Teutonico etal., 1991). Utilization of numerous secondary metabolites in cropprotection, either by conventional plant breeding or by geneticengineering, is currently being evaluated (Gatehouse et al., 1992;Hallahan, supra).

Thus, although phytohormones, and cytokinins in particular, have beenimplicated in conferring resistance to insects in plants, the role ofcytokinins in such resistance has not heretofore been evaluated nor haveany attempts been made to utilize endogenous cytokinins to conferincreased resistance to insects in plants.

SUMMARY OF THE INVENTION

We have discovered that expression in plants of a bacterial geneencoding the first enzyme in the cytokinin biosynthetic pathway,isopentenyl transferase (ipt), reconstructed to allow for woundregulated expression in plants, confers enhanced resistance to insectattack. The ipt gene was fused to a promoter (control region) from apotato gene originally isolated from wounded tubers. Expression of thereconstructed gene was demonstrated in leaves of transgenic plantsfollowing mechanical wounding or insect feeding.

In accordance with this discovery, it is an object of the invention toprovide a gene construct comprising a wound inducible promoter regionand a DNA sequence encoding the enzyme isopentenyl transferase.

It is also an object of the invention to provide a transformation vectorcomprising the novel gene construct subcloned into a vector effectivefor introducing the gene construct into a plant.

It is another object of the invention to provide a transgenic planthaving enhanced insect resistance, wherein the enhanced resistance is aresult of expression of the novel gene construct.

Other objects and advantages will become readily apparent from theensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence of 3' end of promoter region of potato PI-IIK gene.

FIG. 2. (A) Sequence of ipt gene. (B) Sequence of 5' region of truncatedgene.

FIG. 3. (A) Chimeric PI-II-ipt gene construct. The potato proteinaseinhibitor IIK gene promoter (PI-II_(p)) was fused through its5'-untranslated region to the coding region of the isopentenyltransferase gene (ipt) from pTiB6S3. A transcription initiation site islocated 101 bp upstream of the ipt gene start codon. (B) Binary Tiplasmid vector (pPICkn62). The vector contains the T-DNA right (TR) andleft (TL) border sequences from pTiT37 for integration into the plantgenome. The NPTII gene is used as a selectable marker forkanamycin-resistant plant cells. The tetracycline resistance gene (TC)allows selection in bacteria. Transcription terminator (rbcS3') is fromthe pea rbcS-E9 gene. (C) Analysis of ipt transcript levels in excisedleaf disks at 6, 24, and 48 hours. Leaf disks were excised from fullyexpanded leaves on preflowering (PF) and flowering (F) homozygousPI-II-ipt plants (102, 108) and control plants. The 0 time correspondsto RNA from tissues collected immediately after excision.

FIG. 4. Percent of mean leaf disk area consumed by larvae feeding onPI-II-ipt, transgenic control (C), and normal (N) plants in 48 hours.Disks were excised from homozygous R2 (102 and 108) and heterozygous RI(57-1, 110, 3-2 and 108) PI-II-ipt plants. Three plates per treatmentwere used in Expt. 1 and 5 plates in Expt. 2 and 3.

FIG. 5. Whole leaf assay for insect resistance. (A) Detached leaves fromflowering, homozygous PI-II-ipt plant 102 (left) and transgenic controlplant (right) 72 hours after infestation with third instar M. sextalarvae. (B) Mean leaf area consumed by larvae after 72 hours of feedingon flowering PI-II-ipt (102, 108), control (C) and normal (N) leaves.Ten leaves per treatment were used. (C) Mean weight gain of larvaefeeding on the same leaves.

FIG. 6. Enhanced resistance of PI-II-ipt plants (left) to M. sextalarvae as compared to transgenic control plants (right). After 19 daysof feeding, the larvae consumed much less of the leaf material onflowering, homozygous 108 plants in comparison to control plants.

DETAILED DESCRIPTION OF THE INVENTION

Current interests are focusing on the combined effects of naturaldefense mechanisms of plants and biotechnology for crop improvement. Therole of cytokinins in insect resistance was therefore evaluated withrespect to their influence on conferring enhanced insect resistance toplants susceptible to insect infestation.

In order to avoid uptake and metabolism associated with exogenoushormone applications, plants were genetically engineered with awound-inducible cytokinin biosynthesis gene, the isopentenyl transferase(ipt) gene isolated from Agrobacterium tumefaciens (A. tumefaciens).While any effective wound-inducible promoter is acceptable, fusion ofthe ipt gene with a promoter from the potato proteinase inhibitor II(PI-II) gene known to be induced in the leaves of transgenic plants bymechanical wounding and/or insect chewing is preferred. The chimericgene was introduced into plants for expression in tissues such asleaves.

The promoter was obtained from the 5' regulatory region of the potatoPI-IIK gene (described by Thornburg et al., 1987, and hereinincorporated by reference). A partial sequence from the 3' end of thepromoter is shown in FIG. 1.

The gene was obtained from a library of potato genes usingnick-translated tomato inhibitor II as a probe. A vector comprisingregulatory regions, both promoter and terminator, of the wound-induciblegene fused to the open reading frame of the CAT gene was constructed andutilized to transform tobacco plants. Expression of the CAT gene inwounded tobacco leaves demonstrated the effectiveness of wound-induciblecontrol on gene expression.

Fragments of the PI-IIK gene containing the promoter and terminatorregions of the gene were inserted into the plasmid pUC13, resulting inplasmid pRT24. A 0.8 kb EcoRI/BamHI DNA fragment containing only thepromoter region of the PI-IIK gene was then obtained from pRT24 forfusion with the ipt gene to form the gene construct.

The ipt gene was cloned from a tumor-inducing plasmid (pTiB6S3,described by van Larebeke et al., 1974, and Barker et al., 1983, bothherein incorporated by reference) carried by A. tumefaciens as describedby Smigocki, 1991, and Smigocki and Owens, 1988 (both hereinincorporated by reference). The ipt gene sequence is shown in FIG. 2A. A7.3 kb EcoRI fragment from the T-DNA region of pTiB6S3 was then clonedinto pBR325. From that fragment, a 1.3 kb ipt-containing fragment wascloned into pUC18. The promoter region was removed with BAL-31exonuclease, and the 5' region of the truncated gene was sequenced (seeFIG. 2B). The sequence contains 723 nucleotides which code for a productof approximately 27 kd. The ipt gene sequence begins at position 8771(ATG) and terminates at position 9493 (TAG) of the T-DNA gene (Barker,supra).

The 0.8 kb fragment containing the PI-IIK promoter was fused byconventional means through its 5' untranslated region to the codingregion of the truncated ipt gene (FIG. 3A). The truncated ipt genecarries its own transcription terminator and polyadenylation signals.Within the reconstructed gene, a transcription initiation site islocated 101 bp upstream of the ipt gene start codon.

An EcoRI/HindIII PI-II-ipt fragment may be subcloned into any vectoreffective for introducing the gene construct into the plant. Vectorseffective for this purpose are pBI221, pGMVNEO, pUC19, pCMC1100 andpDG208. In a preferred embodiment, the fragment was subcloned into abinary plant transformation vector and mobilized into A. tumefaciens forinfection and transformation of leaf disks (FIG. 3B). Binary planttransformation vectors are known in the art, and selection of aneffective vector is well within the level of skill in the art. Examplesof useful vectors are pEND4K, pMON120, pMON200, pGA472, pKYOX4, pKYOX5,pBIN6, pBIN19, pAGS112, pAGS113, and pKYLX71. Preferred vectors arepKYOX4, pKYOX5, pBIN6, pBIN19, pAGS113 and pKYLX71, while particularlypreferred is pKYLX71.

The ipt gene constructs were transferred to the plant genome byco-cultivation of A. tumefaciens with leaf disks. The leaf pieces may bepreincubated on agar media for 1-2 days prior to infection with thebacteria to enhance transformation. After co-cultivation, leaf pieceswere washed with media and plated on selective agar media containingkanamycin. Cefotaxime and carbenicillin are added to the wash media andselective agar media to kill all bacteria.

Kanamycin-resistant transformed shoots were regenerated, and shoots weretransferred to fresh media and rooted in the presence of kanamycinsulfate. Seeds from the primary transformants were germinated onkanamycin-containing media and resistant progeny screened for woundinducible expression of the ipt gene.

The ipt gene transcript levels in leaf disks excised from fully expandedleaves of preflowering and flowering PI-II-ipt increased approximately25- to 35- fold in 24 hrs. About 50% of the transcripts were stilldetected after 48 hrs (FIG. 3C). At all time points analyzed, the iptmessage levels in flowering plants were 2- to 5- fold higher than inpreflowering plants.

The novel construct is useful for conferring enhanced insect resistanceto a wide variety of plants. Agricultural crop plants are of particularimportance because of their susceptibility to insect infestation and theneed to reduce the amount of applications of chemical insecticides.Other plant types are contemplated, however, including fruit trees suchas peach, plum, etc. as well as ornamental plants which are alsosusceptible to insects.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims.

EXAMPLES Example 1

A chimeric cytokinin gene was constructed by fusing the bacterial iptgene to the 5' regulatory region of the potato PI-IIK gene as describedsupra. The 0.8 kb EcoRI/BamHI fragment was fused through its 5'untranslated region to the coding region of the ipt gene from pTiB6S3.An EcoRI/HindIII pPI-II-ipt fragment was subcloned into a binary planttransformation vector pKYLX71 and mobilized into A. tumefaciens strainEHA101 (pEHA101) for infection and transformation of Nicotianaplumbaginifolia (N. plumbaginifolia) leaf disks. A binary vectorcarrying a truncated ipt (t-ipt) gene without a functional promoter wasused as a negative control for transformation experiments.

Example 2

Reconstructed ipt genes were transferred to the plant genome bycocultivation of A. tumefaciens with N. plumbaginifolia leaf disks.Leaves from 6- to 10-week old sterile plants were cut into approximately4-mm×4-mm pieces and preincubated on agar media for 1-2 days prior toinfection with the bacteria to enhance transformation. After a 24-hrcocultivation, the leaf pieces were washed with media containingCefotaxime and carbenicillin (200 μg/ml, Sigma) and plated on selectiveagar media containing kanamycin (100 μg/ml) as well as the Cefotaximeand carbenicillin.

Ten kanamycin-resistant, independently transformed shoots wereregenerated. Seeds from the primary transformants were germinated onkanamycin-containing media (100 μg/ml) and resistant progeny screenedfor wound inducible expression of the ipt gene. Three 1-in incisionswere made on each half of a fully expanded leaf, and 24 hr later RNA wasextracted and analyzed. Homozygous kanamycin-resistant R2 progeny wereselected from R1 plants segregating 3:1 for kanamycin resistance.

Example 3

Fully expanded leaves on PI-II-ipt plants were used in leaf disk andwhole leaf assays. Leaf disks 1.45 cm² were cut with a cork borer andplaced in a 60×15 mm petri dish on filter paper wet with water or waterplus the cytokinin zeatin at 10 and 20 μg/ml in 1 or 2% methanol,respectively (Sigma). Petioles of detached leaves were submerged insealed vials containing water or water plus zeatin as above and placedin large petri dishes lined with filter paper. Surface areas of leafdisks and leaves were measured before and after insect feeding with asurface area meter (LI-COR, Inc., Lincoln, Nebr.). Data in FIGS. 4 and 5was analyzed by analysis of variance and means were compared using theleast significant difference test.

Manduca sexta (M. sexta) larvae (tobacco hornworms) were maintained on ameridic diet prior to feeding trials. For each experiment, a weighedneonate or a third-instar larvae was placed in a petri dish with either5 leaf disks or a whole leaf. The larvae were allowed to feed for 48 or72 hours. When whole plants were infested, a single neonate larvae wasused per plant, and plants were wrapped during the last instar withcheesecloth to prevent escape. Larval weights were recorded weekly.Three to five plants per treatment were infested in 3 replicatedexperiments. Results are shown in FIG. 4. Larvae consumed only 8 and 13%of the leaf disks as measured by area when feeding on homozygoustransformants 102 and 108, respectively (Experiment 1). In comparison,larvae fed disks excised from transgenic control and untransformedplants consumed 5-11 times more of the tissues. Insects feeding on disksfrom heterozygous R1 plants (57-1, 110, 3-2, 108) consumed approximately20 to 50% less than those feeding on controls.

Two additional experiments with heterozygous plants 56-1 and 100confirmed a significant decrease in leaf disk area consumption whencompared to the controls. An overall reduction in all disks consumed bythe larva in Experiments 2 and 3 corresponds to less feeding by neonatesvs. third instar larvae used in Experiment 1. The specific leaf weight(g/cm²) of the PI-II-ipt plants is reduced by about 40%, therebyincreasing the significance of the observed enhanced resistance.Homozygous R2 progeny were further analyzed by an excised leaf assay(FIG. 5A). Third-instar tobacco hornworm larvae feeding on leaves fromflowering (PI-II-ipt plants (102 and 108) consumed only about a third toa half of what was consumed by larvae feeding on control leaves (FIG.5B; Table 1). Mean larval weight gain was reduced by approximately 30 to60% compared to controls FIG. 5C; Table 1).

Resistance at the whole plant level was evaluated by infestinghomozygous PI-II-ipt flowering plants with a single neonate hornwormlarvae. Approximately 19 days later, the larvae pupated, however, theconsumption of the PI-II-ipt plants was greatly reduced in comparison tothe control plants (FIG. 6). In general, all the insects preferred thenewly emerging and younger leaves, and only when these were exhausteddid they feed on the older leaves. During the course of the experiment,weights of larvae feeding on PI-II-ipt or control plants did not differsignificantly and all emerging adults appeared normal.

Myzus persicae (M. persicae), green peach aphid, nymphs were propagatedin a rearing chamber for feeding experiments to evaluate the resistanceof PI-II-ipt plants. A nymph less than 12 hr old was placed on a singleleaf disk. After 8 days, it was determined whether the nymph was aliveor dead, the stage of development (nymph or adult female) and whether ornot the adult female had reproduced. Results are presented in Table 2.

After 8 days of feeding on disks from flowering PI-II-ipt plants (102and 108), approximately 30 to 40% of the nymphs developed into adultfemales. Of those adults, 50 to 80% reproduced. On control and normaltissues, on the average, 74% of the nymphs reached adulthood, and 93%reproduced.

Feeding zeatin through petioles of leaves excised from PI-II-ipt plantprior to flower development, caused about a 40 to 60% reduction in meanarea consumed by the tobacco hornworm larvae and a 30% reduction of meanlarval weight gain as compared to normal controls (Table 1). Zeatinuptake by leaves from flowering PI-II-ipt plants boosted the level ofinsect resistance normally observed in these plants by up to 36% basedon mean area consumed. A similar response to zeatin was not observedwith any of the leaves from normal, untransformed plants. In controlexperiments with 1% methanol alone, no negative effects on the hornwormfeeding were observed.

Placing leaf disks from PI-II-ipt or control and normal plants on filterpaper wet with zeatin at 10 or 20 μg/ml delayed the development of thegreen peach aphid nymphs (Table 2). Of the surviving nymphs, most wereimmature and did not reproduce within the 8 day test. Nymphs feeding oncontrol leaf disks placed in 1 or 2% methanol developed normally.

Table 1. Resistance of homozygous R2 PI-II-ipt plants (102, 108) to thetobacco hornworm larvae. Effects of zeatin (Z; 10 μg/ml) were determinedby calculating the mean leaf area consumed and larval weight gain after3 days of feeding. Each number represents an average of six replicates.

                  TABLE 1                                                         ______________________________________                                        Mean leaf area consumed (cm.sup.2) Mean larval weight gain (g)                Preflowering      Flowering   Preflowering                                                                             Flowering                            Plants --     Z       --  Z     --   Z     --  Z                              ______________________________________                                        102    25      9      18   5    .23  .22   .17 .10                            108    24     14      14  11    .25  .23   .23 .17                            normal 24     23      34  29    .29  .32   .32 .30                            ______________________________________                                    

Table 2. Results of green peach aphid feedings on flowering homozygousR2 PI-II-ipt plants (102, 108) without and with exogenously suppliedzeatin (Z; 10 μg/ml and 20 μg/ml). Presented are the percent of nymphsstill alive and the percent of adult females (if any) that hadreproduced at the end of the 8 day test. In tests without added zeatin,each number represents an average of four independent tests done inreplicates of 10 disks per treatment. The zeatin results are averages oftwo independent tests with 5 disks per treatment.

                  TABLE 2                                                         ______________________________________                                        Percent alive   Percent adult females with nymphs                             Zeatin (μg)  Zeatin (μg)                                                Plants  0       10    20  0        10     20                                  ______________________________________                                        102     40      10    10  53       0      0                                   108     32      50    10  83       50     0                                   control 67      20    20  89       0      0                                   normal  80      20    60  97       50     40                                  ______________________________________                                    

Experiment 4

The concentration of zeatin and N⁹ substituted zeatin derivatives, majorcytokinins produced in tissues transformed with the PI-II-ipt gene, weredetermined using analytical kits (De Danske Sukkerfabrikker, Copenhagen;IDETEK, Inc., San Bruno, Calif.). Plant tissues were extracted in 80%methanol overnight at -80° C. All extracts were purified on columnspacked with anti-zeatinriboside antibodies, and eluted cytokinins werequantified by ELISA. To determine the percent recovery, control sampleswere spiked with 1000 to 2000 pmoles of zeatinriboside or zeatin(Sigma). For each plant, 3 to 4 samples were analyzed.

Levels of zeatin and zeatinriboside cytokinins were greatly elevated. Onthe average, zeatin and zeatinriboside concentrations increased toapproximately 500 to 550 pmoles/g fresh tissue. This corresponds to agreater than 70-fold increase over endogenous cytokinin levels incontrol tissues. Cytokinin levels in detached leaves at the end of a72-hr infestation were slightly lower (approximately 400 pmoles/g).

DISCUSSION

When the M. sexta larvae were fed leaf disks or whole leaves fromflowering PI-II-ipt plants, they consumed significantly less of theplant material than larvae feeding on leaves from control plants (FIG. 4and 5; Table 1). A corresponding decrease in larval weight gain was alsoobserved. At the whole plant level, less PI-II-ipt leaves were consumedbut no significant differences in larval weights were recorded (FIG. 6).It appears that sufficient feeding material is provided by youngerleaves and the abundance of lateral buds released during reproductivestage of growth of the PI-II-ipt plants (Smigocki, unpublished). Onnormal plants, newly emerging and younger leaves have been reported tobe preferred by these insects (Thornburg, supra). We find lower ipttranscript levels and cytokinin concentrations in younger leaves oftransgenic PI-II-ipt plants (FIG. 3).

Green peach aphid feedings on leaf material from flowering PI-II-iptplants delayed normal development of newly hatched nymphs into adultfemales by about 50% (Table 2). In addition, of the nymphs that reachedmaturity, fewer were able to reproduce as compared to controls. Enhancedresistance to the tobacco hornworm and green peach aphid is observedwhen PI-II-ipt plants are in the mid to late flowering stage of growth(Table 1).

Cytokinin levels were elevated by approximately 70-fold in comparison tocontrols. By boosting the endogenous cytokinin levels with exogenousapplications of zeatin, enhanced resistance to the tobacco hornworm wasinduced in leaves from preflowering plants (Table 1). In addition, ahigher degree of resistance was also observed when leaves from floweringPI-II-ipt plants were supplied with zeatin. This response to zeatin wasnot observed with leaves form normal, untransformed plants and mayreflect problems associated with sufficient uptake, metabolism, orcompartmentalization of exogenously supplied cytokinins necessary toretard hornworm feeding. The effects of exogenous zeatin applications ondelaying the green peach aphid development were more dramatic in thatmost of the nymphs did not reach maturity. The green peach aphidtolerance to cytokinin effects appears to be lower than that of thetobacco hornworm and may be directly related to their much reducedoverall body mass. Zeatin application results suggest that use of astronger constitutive promoter to express the cytokinin gene wouldincrease endogenous cytokinin concentrations to even higher levels thanthose in PI-II-ipt plants and result in better insect control. It haspreviously been reported that overexpression of the ipt gene with the35S promoter from cauliflower mosaic virus increases zeatin levels up toseveral hundred fold in N. plumbaginifolia (Smigocki and Owens, 1989).However, the constitutive overproduction of cytokinin in plant cellsinhibits regeneration of whole plants. Temporal and tissue specificexpression allows for regeneration of plants and is preferred forexpression of a foreign gene as for example in leaves upon insectfeeding.

We claim:
 1. A transgenic plant having enhanced resistance to insectfeeding on plants, said plant comprising a gene construct comprising awound inducible promoter region fused to a DNA sequence encoding theenzyme isopentenyl transferase.
 2. The transgenic plant of claim 1,wherein the wound inducible promoter of said gene construct is derivedfrom the potato proteinase inhibitor II gene.
 3. The transgenic plant ofclaim 2, wherein said promoter is obtained from the 5' regulatory regionof the potato proteinase IIK gene.
 4. The transgenic plant of claim 1,wherein said DNA sequence is derived from Agrobacterium tumefaciens.